The ribosomal RNA genes (xe2x80x9crRNAxe2x80x9d) of eukaryotic cells are transcribed by an enzyme solely dedicated to that purpose, RNA polymerase I (xe2x80x9cpol Ixe2x80x9d). The expression of the rRNA genes is coordinated with cellular proliferation. When cell growth is impaired by nutrient deprivation or depletion, transcription of the rRNA genes declines: This decline is reversed when growth-permissive conditions are restored. Since growth-rate dependence is a universal feature of rRNA gene regulation, identifying the molecular mechanisms that couple pol I activity to cell growth is a central question in studies of all eukaryotic systems, including the yeast and mammalian systems.
Transcription of rRNA genes of the yeast Saccharomyces cerevisiae requires the activity of at least three transcription factors, which have been defined both genetically and biochemically. Two of these factors, Core Factor and Upstream Activation Factor, are multi-subunit factors that interact directly with distinct elements of the rRNA promoter to assemble a preinitiation complex. Core Factor (xe2x80x9cCFxe2x80x9d) is composed of three essential gene products, Rrn6, Rrn7, and Rrn11, and associates with TATA box binding protein (xe2x80x9cTBPxe2x80x9d). CF is required to direct transcription initiation from the core promoter of an rRNA gene both in vitro and in vivo (see Keys et al., Genes Dev. 8:2349-62 (1994); Lin et al., Mol. Cell. Biol. 16:6436-43 (1996); Steffan et al., Genes Dev. 10:2551-63 (1996); Keener et al., J. Biol. Chem. 273:33795-802 (1998); Lalo et al., J. Biol. Chem. 271:21062-67 (1996)). Upstream Activation Factor (xe2x80x9cUAFxe2x80x9d) binds to the upstream promoter element and stimulates transcription from the core promoter. When the yeast genes encoding the UAF subunits Rrn5, Rrn9, or Rrn10 are individually disrupted, cells remain viable but exhibit pronounced growth defects, indicating that UAF activity is necessary to support levels of rRNA synthesis required for normal cell growth (see Keys et al., Genes Dev. 10:887-903 (1996)). UAF subunits interact with CF subunits in vitro, and direct interaction of UAF with TBP has been shown to mediate transcriptional activation in vivo (see Steffan et al. (1996), supra; Steffan et al., Mol. Cell. Biol. 18:3752-61 (1998)).
A third transcription factor, Rrn3, is unique in that it functions as a single subunit, shows no sequence-specific DNA binding activity, and is not required for pre-initiation complex assembly (see Yamamoto et al., EMBO J. 15:3964-73 (1996)). The Rrn3 protein appears instead to function by direct interaction with RNA polymerase I since it is stably associated with pol I in transcriptionally active extracts (see Milkereit and Tschochner, EMBO J. 17:3692-703 (1998)). The transcriptional activity of pol I is enhanced by pre-incubation with Rrn3 protein in the absence of either DNA template or other pol I transcription factors (see Yamamoto et al. (1996), supra; Keener et al. (1998), supra). The interaction of Rrn3 polypeptide with RNA polymerase I fluctuates with changes in cellular growth rate; Rrn3 polypeptide is not associated with pol I in transcriptionally-inactive extracts prepared from growth-arrested cells. Transcriptional activity is restored upon addition of Rrn3-associated pol I purified from growing cells (see Milkereit and Tschochner, supra). These observations suggest that Rrn3 activity may be regulated in a growth-dependent manner. Although the specific function of Rrn3 polypeptide is as yet unknown, it is essential for rRNA gene transcription in vivo and in vitro, and it may be required to mediate productive interactions of pol I with the pre-initiation complex.
Transcription of mammalian rRNA genes also requires two promoter-binding transcription factors which appear to perform functions similar to those of the yeast pol I factors. The mammalian transcription factors are not conserved with those of yeast. The core promoter-binding factor IF-IB/SL1, which is essential for transcription, is comprised of TBP and three transcription-associated factors (xe2x80x9cTAF""sxe2x80x9d). The TAF""s are similar in size to the three yeast CF subunits, but display no amino acid sequence similarity to their yeast counterparts (see Comai et al., Cell 68:965-76 (1992); Eberhard et al., Nucleic Acids Res. 21:4180-86 (1993); Heix et al., Proc. Natl. Acad. Sci. USA 94:1733-38 (1997)). The upstream stimulatory activity in mammalian cells is mediated by a single protein, UBF, which bears no resemblance to the multi-subunit yeast UAF complex (see Bell et al., Science 241:1192-97 (1988); Schnapp and Grummt, J. Biol. Chem. 266:24588-95 (1991)). DNA binding by UBF is mediated by high mobility group domains which are not present in any of the yeast pol I transcription factors, and no proteins related to the yeast UAF subunits have been identified in mammals to date. It therefore appears that the promoter-binding factors of yeast and mammals are evolutionarily divergent.
In mammalian cells, two RNA polymerase I-associated factors, TIF-IA and TIF-IC, have been identified (see Schnapp et al., EMBO J. 9:2857-63 (1990); Schnapp et al., EMBO J. 13:4028-35 (1994)). Like yeast Rrn3 (xe2x80x9cyRrn3xe2x80x9d), TIF-IA and TIF-IC are not required for pre-initiation complex assembly but are essential for transcription initiation by pol I (see Schnapp and Grummt, supra). The relationship of these factors to yRrn3 has not yet been determined since their genes have not yet been isolated. However, TIF-IA shares an important functional similarity with yRrn3 in that its activity is regulated by cellular growth rate (see Schnapp et al. (1990), supra; Schnapp et al., Mol Cell. Biol. 13:6723-32 (1993)).
rRNA synthesis is required for cell division and differentiation, and also for normal cellular metabolism. The assembly of new ribosomal subunits requires new rRNA transcripts. Thus, rRNA transcription provides a common regulatory point for controlling cell proliferation in a wide variety of cell types. Reagents that modulate rRNA transcription could be used to affect proliferation in such a wide variety of cell types, and would not be limited to current reagents that are cell-type specific. For example, reagents, and methods of their use, that modulate rRNA synthesis could be used to stimulate hypoproliferative cells or to inhibit hyperproliferative cells, in diseases such as cancer. There is need, therefore, for reagents, and methods of using such reagents, to modulate cell proliferation through rRNA transcription. Surprisingly, the present invention fulfills these and other related needs.
The present invention relates to the discovery, identification and characterization of eukaryotic RRN3 genes. The invention encompasses nucleotide sequences of the RRN3 gene and amino acid sequences of its encoded polypeptide product, as well as fragments, derivatives and analogs thereof. The invention also encompasses the production of Rrn3 polypeptides and antigen-specific antibodies. The invention further encompasses compositions and methods for screening, diagnostic and therapeutic applications.
One aspect relates to RRN3 nucleic acids, including mRNAs, DNAs, cDNAs, genomic DNA, as well as RRN3 antisense nucleic acids. Such nucleic acids include the RRN3 cDNA having the nucleotide sequence of SEQ ID NO:1. Another aspect relates to RRN3 nucleic acid derivatives or fragments that encode Rrn3 polypeptides, or portions thereof. Such derivatives include nucleic acids encoding all possible codon choices for the same amino acid or conservative amino acid substitutions thereof. Other RRN3 nucleic acids include those nucleic acids that are capable of selectively hybridizing to a human RRN3 cDNA (e.g., SEQ ID NO:1) under stringent hybridization conditions. A related aspect of the present invention relates to nucleic acid probes comprising polynucleotides of sufficient length to selectively hybridize to a polynucleotide encoding an Rrn3 polypeptide of the present invention.
In another aspect, the present invention provides substantially pure preparations of human Rrn3 and polypeptide fragments, derivatives and analogs thereof. In a related aspect, the invention concerns nucleic acid constructs for expressing the RRN3 nucleic acids. Such expression constructs typically comprise a transcriptional promoter, a nucleic acid which encodes the Rrn3 polypeptide, derivative or fragment thereof, and a transcriptional terminator, each operably linked for expression of the Rrn3 polypeptide, derivative or fragment. In another aspect, the invention provides the ability to produce Rrn3 polypeptides, derivatives, or fragments thereof by recombinant means, typically in cultured eukaryotic cells or in prokaryotic cells. The expressed Rrn3 polypeptide, derivatives or fragments can have the same functional activity as the corresponding native Rrn3 polypeptide, or an altered activity. Accordingly, isolated and purified polynucleotides are described which encode Rrn3 polypeptide, derivatives, and fragments thereof, where the polynucleotides can be in the form of RRN3 nucleic acids, such as genomic DNA, cDNA, or mRNA.
In another aspect, the RRN3 nucleic acids can be used to identify other mammalian genes that encode Rrn3-like molecules. The RRN3 nucleic acids can also be used to screen for mutations in a RRN3 gene that are associated with certain diseases. As such, the invention further relates to materials and methods for the identification of disease-associated mutations, where the RRN3 nucleic acids are used to detect the presence of mutations in a biological sample. RRN3 nucleic acid probes can also be used to identify mutations in a RRN3 gene for diagnostic purposes. The probes can be full-length genomic DNA, cDNA, RNA or nucleic acids as small as from about 14 to 25 nucleotides, more often though from about 40 to about 50, or more nucleotides in length.
The invention also provides antibodies to Rrn3 polypeptide, in the form of polyclonal or monoclonal antibodies. Such antibodies can specifically bind to an Rrn3 polypeptide or fragment, derivative or analog thereof, and can be incubated with a biological sample under conditions conducive to immune complex formation, such as by ELISA. The resulting complexes can then be detected, typically by means of a label such as an enzyme, fluorophore, radionuclide, chemiluminescent, particle, or a second labeled antibody. Thus, means are provided for immunohistochemical staining of tissues, including tumor biopsies. The invention further relates to methods of identifying agonists and antagonists that modulate the activity of an Rrn3 polypeptide, and further provides a method for identifying agents that specifically affect one Rrn3 polypeptide, without affecting other Rrn3 polypeptides.
A further understanding of the nature and advantages on the invention will become apparent by reference to the remaining portions of the specification.